Therapeutic agent for ischemia which inhibits apoptosis under ischemic condition

ABSTRACT

The present invention relates to a therapeutic agent for ischemia which inhibits apoptosis under ischemic condition. The therapeutic agent of the present invention comprises antibacterial agents of quinolones, quinones, aminoglycosides or chloramphenicol as an active ingredient. Since the invented therapeutic agent improved the viability of cells under hypoxic and hypoglycemic condition, it can be clinically for ischemic diseases such as applied as a potential drug for ischemia-associated infarction and cerebral infarction.

CROSS-REFERENCE TO RELATED APPLICATIONS

This is the U.S. National Phase under 35 U.S.C. §371 of InternationalApplication No. PCT/KR01/00050, fired Jan. 12, 2001 and published inEnglish, which claims priority to Korean Application No. 2000/1309,filed Jan. 12, 2000.

BACKGROUND OF THE INVENTION

1. Field of the Invention

The present invention relates to a therapeutic agent for ischemia, morespecifically, to a therapeutic agent for ischemia which inhibitsapoptosis under ischemic condition.

2. Description of the Prior Art

As the death rate from cardiovascular diseases is increasing recently,researches on the cardiovascular diseases are now in rapid progress.Among them, one of the most noticeable field is that relating tothrombus, wherein efforts to restore blood vessel functions bydissolving thrombus which is the major cause of blockage of bloodvessels, and, furthermore, to inhibit thrombus formation are being made.However, there is little progress in developing method for preventingripple effects of blockage of blood vessels caused by thrombus or othercauses. Accordingly, in case of a patient dying of the blockage of bloodvessels, it is almost impossible to alleviate ischemic injury whichlacks an adequate supply of oxygen and glucose.

It has been reported that administration of antibiotics to the patientwho has antibody against Chlamydia pneumoniae which is related to onsetof acute myocardial infarction reduces the onset rate of acutemyocardial infarction(see: Meier C. R. et al., JAMA, 281(5):427-431,1999). Antibiotics such as quinolones or quinones could reduce onsetrate of acute myocardial infarction, on the other hand, antibiotics ofmacrolide which are known to be the most effective agents to killChlamydia pneumoniae have no effect on reducing the onset rate of acutemyocardial infarction, suggesting that the antibiotics are not merelykilling pathogenic microorganisms. Thus, antibiotics have been regardedas thrombosis inhibitors or thrombolytic agents, however, there is noevidence of relations between antibiotics and thrombus, hence,antibiotics can be conjectured to work on acute myocardial infarctionvia other mechanism than involvement of thrombus. The fact thatantibiotics exert a certain effect on acute myocardial infarctionwithout involvement of thrombus implies that antibiotics may protectcells from destruction caused by inadequate supply of oxygen and glucosedue to the blockage of blood vessels. Accordingly, it could be expectedthat the patient who has ischemia due to the blockage of blood vesselcan be recovered by using antibiotics, however, there is still littleprogress in researches of this area.

Under the circumstances, there is a continuing need to understand themechanism of inhibiting cell damage which is prelude to ischemia and todevelop an agent effectively inhibiting the ischemic cell damage.

SUMMARY OF THE INVENTION

The present inventors have made an effort to elucidate the mechanism ofinhibiting cell damage which is prelude to ischemia and to develop atherapeutic agent effectively inhibiting the said ischemic cell damage,and, based on the fact that the death of cells under hypoxic andhypoglycemic condition is progressed via apoptosis, they discovered thatthe addition of antibiotics of quinolones, quinones, aminoglycosides orchloramphenicol to the cells under hypoxic and hypoglycemic conditioncan dramatically inhibit apoptosis, furthermore, the apoptosis can beinhibited by administering the antibiotics to an individual underischemic condition in an amount of administering to the individualinfected with pathogenic microorganisms.

A primary object of the present invention is, therefore, to provide atherapeutic agent which inhibits apoptosis under ischemic condition.

BRIEF DESCRIPTION OF THE DRAWINGS

The above and the other objects and features of the present inventionwill become apparent from the following descriptions given inconjunction with the accompanying drawings, in which:

FIG. 1 is a graph showing HepG2 cell viability under various oxygenconditions.

FIG. 2a is a graph showing the dependency of HepG2 cell viability onglucose concentration with incubation time under a low oxygen condition.

FIG. 2b is a graph showing the change in residual glucose concentrationwith time depending on initial glucose concentration under a low oxygencondition.

FIG. 2c a graph showing the change in pH with time depending on glucoseconcentration under a low oxygen condition.

FIG. 3 is a graph showing HepG2 cell viability at various geneticinconcentrations.

FIG. 4 is a graph showing cell viability of HepG2 treated with variousantibiotics.

DETAILED DESCRIPTION OF THE INVENTION

The therapeutic agent of the invention, which inhibits apoptosis underischemic condition, comprises antibiotics of quinolones, quinones,aminoglycosides or chloramphenicol as active ingredients

The present inventors, first of all, examined that apoptosis is inducedin cells under ischemic condition which lacks an adequate flow of bloodto supply oxygen and glucose due to blockage of blood vessels bythrombus or other causes, and acknowledged that glucose generates energyvia TCA cycle and electron transfer system in the presence of oxygen,however, under hypoxic (low oxygen) condition, glucose is converted tolactic acid which generates only a little energy, and resumes energygeneration if sufficient oxygen is supplied.

In order to simulate ischemic cells which lack the supply of oxygen andglucose due to the blockage of blood vessels by thrombus or othercauses, the present inventors created ischemic condition bydiscontinuing supply of oxygen and glucose to the cultured cells, andthen observed the changes occurred in the cells. When oxygen wasdepleted in the cells, glucose also became depleted and the cells weredied without utilization of lactic acid. When sufficient oxygen wassupplied and glucose became depleted, the cells could survive untillactic acid was used up, that is, cells died with exhaustion of lacticacid. However, when cells were treated with antibiotics of quinolones,quinones, aminoglycosides or chloramphenicol, it was found that thecells were viable for a certain period of time even after exhaustion ofglucose and production of lactic acid. Preferably, the antibioticsinclude, but are not intended to be limited to, quinolones of 10-100μg/ml levofloxacin, 10-100 μg/ml ofloxacin or 1-10 μg/ml ciprofloxacin;quinones of tetracycline, minocycline, doxycycline, or oxytetracyclineat a of 0.1-10 μg/ml each; and, aminoglycosides of 10-100 μg/mlgeneticin, 500-1000 μg/ml neomycin or 100-1000 μg/ml gentamycin. In caseof chloramphenicol, a concentration of 1-10 μg/ml were preferablyemployed.

Analyses of various test groups of cells under a condition of oxygen andglucose depletion have shown that the groups of cells without antibiotictreatment underwent typical apoptosis, whereas, the groups of cellstreated with said antibiotics did not undergo apoptosis for a certainperiod of time. These results imply that the said antibiotics inhibitapoptosis occurred in cells with ischemic injury which lacks an adequatesupply of oxygen and glucose. Additional experiments demonstrated thatantibiotics somehow affect the expression of bcl-2 protein which isknown to be an inhibitor of apoptosis in cells with ischemic injury.

In order to examine if the results obtained with cultured cells in vitrocan be applied to the tissue with ischemic injury, the rats underischemic condition were treated with the said antibiotics and thenhearts from the rats with or without antibiotic treatment were subjectto biopsy, and found that the preservation rate of cardiac tissues fromrats treated with the antibiotics was higher than that withoutantibiotic treatment. Accordingly, it was clearly demonstrated that thetherapeutic agent comprising active ingredients of antibiotics such asquinolones, quinones, aminoglycosides or chloramphenicol improves theviability of cells under hypoxic and hypoglycemic condition, assuringthat it can be clinically applied as a potential drug forischemia-associated diseases such as myocardial infarction and cerebralinfarction.

Considering that most of experimental data using rats intend to apply tomammals and antibiotics employed in the invention are commonlyadministered to human bodies, the therapeutic agent of the invention,which is prepared on the basis of the experimental results, may beapplied to human body to inhibit apoptosis induced under ischemiccondition.

The present invention is further illustrated in the following examples,which should not be taken to limit the scope of the invention.

Example 1: Cell viability under various oxygen conditions

HepG2 cells (human hepatoma cell line, ATCC HB 8065, 1×10⁶cells/60 mmculture dish) were grown in a minimal essential medium supplemented with100unit/ml penicillin, 100 μg/ml streptomycin, 1 g/l glucose, 2.2 g/lsodium bicarbonate, and 10% (w/v) fetal calf serum for 2 days, followedby feeding with the same medium and incubating under an environment of1, 2, or 5% (v/v) oxygen, respectively. Numbers of viable cells withtime were determined by trypan blue exclusion assay using hemocytometerafter 10-15 minutes of incubation of 1:1 (v/v) mixture of 0.4% (w/v)trypan blue and cell suspension. Cell viability with time wasrepresented in the ratio of viable cell number to cell number justincubation condition was changed to a low oxygen condition(see: FIG. 1).FIG. 1 is a graph showing cell viability under various oxygenconditions, where () indicates 1% (v/v), (▾) indicates 2% (v/v), (▪)indicates 5% (v/v), and (♦) indicates 21% (v/v) oxygen, respectively. Asshown in FIG. 1, it was clearly demonstrated that HepG2 cells wereviable in a minimal medium containing low concentration of glucose underan environment over 5% (v/v) oxygen, whereas, the cells died under anenvironment of less than 2% (v/v) oxygen. Accordingly, a low oxygencondition was set at 1% (v/v) oxygen in the following examples.

Example 2:Cell viability depending on glucose concentration HepG2 cellswere cultured analogously as in Example 1 except for 1% (v/v) oxygen andvaried glucose concentrations from 1 to 4.5 g/L. Then, cell viability,changes in glucose concentration and changes in pH with time weremeasured, respectively(see: FIGS. 2a, 2 b and 2 c). FIG. 2a shows cellviability with culture time, 2 b shows changes in glucose concentrationwith time, and 2 c shows changes in pH with time, where () indicates 1g/L glucose, (◯) indicates 2 g/L glucose, (▾) indicates 3 g/L glucose,(∇) indicates 3.5 g/L glucose, (▾) indicates 4 g/L glucose, and (□)indicates 4.5 g/L glucose, respectively. As shown in FIGS. 2a-2 c, itwas demonstrated that cells were died as a result of depletion ofglucose or lowering of pH under a low oxygen condition.

Example 3: Cell viability under various geneticin concentrations

HepG2 cells were cultured for 2 days in the same manner as in Example 1,and then, the maximum concentration of geneticin at which HepG2 cellscan survive was determined by replacing the culture medium with a freshmedium containing 0-1000 μg/ml geneticin, an aminoglycoside antibioticunder an environment of 1%(v/v) oxygen, respectively(see: FIG. 3). FIG.3 is a graph showing the cell viability at various geneticinconcentrations, where geneticin was added at a concentration of 0 μg/ml(), 1 μg/ml (◯), 3 μg/ml (▾), 10 μg/ml (∇), 100 μg/ml (▪), and 1000 μg/ml (□), respectively. As shown in FIG. 3, it was clealy demonstratedthat cells treated with 10-100 μg/ml genticin were viable for a certainperiod of time under an environment of 1% (v/v) oxygen.

Example 4: Effects of various antibiotics on cell viability

In order to screen antibiotics which have similar effect to geneticinbut have different chemical structure, HepG2(Human hepatoma cell line,ATCC HB 8065) cells were grown under the same condition described inExample 1, followed by replacing the medium with fresh medium proper fortest conditions described below, and then, cell viabilities undervarious conditions were compared after 2 days of incubation. Test groupswere divided as follows depending on test conditions: test group A with21% (v/v) oxygen and 4.5/L glucose, test group B with 21(v/v) oxygen and1 g/L glucose, test group C with 1% (v/v) oxygen and 4.5/L glucose, testgroup D with 1%(v/v) oxygen and 1 g/L glucose, test group E withaminoglycoside antibiotic of geneticin (10 μg/ml) treated test group D,test group F with quinolone antibiotic of ofloxacin test group D, andtest group G with antibiotic of doxycycline (0.1 μg/ml) treated testgroup D (see: FIG. 4). FIG. 4 is a graph showing the comparison ofeffects of various antibiotics on the cell viability. As shown in FIG.4, it has been found that: cell viability of test group D was lowcompared to test groups A, B and C, and cell viability of test group Dcan be recovered by treatment of various antibiotics.

Example 5: Effect of antibiotics on ischemic cells

Male Sprague-Dawley rats weighing 250 g were injected peritoneally with45 mg/kg geneticin in test group 1, with 45 mg/kg doxycycline in testgroup 2, and with 1 cc saline in a conrol group, respectively. Ten ratswere included in each test group. After rats were anesthesized byintramuscular injection with 75 mg/kg ketamine and 5 mg/kg xylazine, atubing was inserted into the airway of the rat and breathing wascontrolled mechanically at a rate of 70-80 breaths per minute andbreathing volume of 15-20 ml/kg. After cutting off the chest bone in themiddle, left anterior descending artery (LAD) was ligatured with 6-0ligature by tying the lower part of the first diagonal branch artery,and the ligature of blood vessel was confirmed by observation ofwhitening cardiac muscle. Two hours later, blood flow was resumed and 1cc of Evans blue solution (5%, v/v) was slowly injected into the leftventricle of the heart, and then the heart was removed from the bodywhile leaving the aorta, followed by fixing the heart by using 20 cc of1% (v/v) triphenyltetrazolium chloride(TTC).

The sections obtained by transverse cutting of the portion from the tipof the heart to right above the ligatured area into 4 equal parts in a 2mm space were photographed by the aid of flat bad scanner, respectively.The photographs were analyzed and classified into three groups: normalarea (Normal) which was stained blue or dark brown, infarct area(Infarct size) which was not stained, and ischemic area(Area at risk)which was stained red. By employing precalibrated image analyzer (ScionImage version 1.62), each stained area was measured. After calculatingIS/LV (percentage of infarct area (IS) per cross-sectional area of heart(LV)), and AR/LV (percentage of ischemic area (AR) per cross-sectionalarea of heart (LV)), IS/(IS+AR)(percentage of infarct area (IS) per sum(IS+AR) of infarct area (IS) and ischemic area was calculated andcompared with each other (see: Table 1).

TABLE 1 Effect of antibiotics on ischemic cells (%) Test groups IS/LVAR/LV IS/(IS + AR) control 22.7 ± 0.9 33.5 ± 2.7 40.9 ± 2.5 test group 112.5 ± 1.8 40.0 ± 4.1 24.6 ± 2.1 test group 2 16.9 ± 2.5 41.8 ± 4.3 29.3± 4.2

As shown in Table 1 above, it was found that: infarct area (IS) werereduced in ischemic cells treated with geneticin and doxycycline; and,there were little deviations owing to ligatured artery, in a view thatthe ratio of normal area ranged 41.3 to 47.5%.

Example 6: Screening of antibiotics exerting effects on cell viability

In order to examine whether antibiotics with other structures thanaminoglycoside antibiotic of geneticin, can also enhance cell viabilityunder a hypoxic condition, analyses were performed as followings: i.e.,after analysis of antibiotics such as geneticin, neomycin, gentamycin,tetracycline, minocycline, oxytetracycline, doxycycline,chloramphenicol, levofloxacin, ofloxacin, ciprofloxacin, ampicillin,amoxicillin, cephalosporin, erythromycin, suifadiazlne, cyclohexamide,5-fluorouracil, puromycin and trimetazidine in accordance with theprocedure described in Examples 2 and 3, antibiotics which showedenhancement of cell viability under hypoxic condition were selected andtheir effective concentrations were determined, respectively (see: Table2).

TABLE 2 Antibiotics exerting enhancement effects on cell viability andtheir effective concentration Antibiotics Concentration (μg/ml)geneticin 10-100 neomycin 1000 gentamicin 100-1000 tetracycline 0.1-10  minocycline 0.1-10   doxycycline 0.1-10   oxytetracycline 0.1-10  chloramphenicol 1-10 levofloxacin 10-100 ofloxacin 10-100 ciprofloxacin1-10

Effective concentration ranges in Table 2 represent the concentrationranges of antibiotics exerting enhancement effects on HepG2 cellviability under 1%(v/v) oxygen condition. As shown in Table 2 above,among the antibiotics known to act on 30S subunit of ribosome in E.coli,neomycin and gentamycin other than geneticin were effective amongaminoglycoside antibiotics. Also, among the antibiotics known to act on30S subunit of ribosome in E.coli, a quinone antibiotic of tetracyclinewas effective at very low concentration range of 0.1-10 μg/me, andtetracycline derivatives such as minocycline, oxytetracycline anddoxycycline were effective at the same range of low concentration.Meanwhile, among the antibiotics known to act on 50S subunit of ribosomein E.coli, an aromatic antibiotic of chloramphenicol was effective, buta macrolide antibiotic of erythromycin was not effective. Amongquinolone antibiotics known to act on DNA gyrase, all analyzedcompounds, levofloxacin, ofloxacin, and ciprofloxacin were effective.However, antibiotics known to inhibit synthesis of cell wall ofmicroorganisms, such as ampicillin, amoxillin, and cephalosporin did notshow enhancement effect on cell viability. Antibiotics such as asulfadiazine which is known to inhibit dihydropteroate synthetase in thefolic acid metabolism, a cyclohexamide inhibiting protein synthesis ineukaryotes, a 5-fluorouracil blocking DNA synthesis by competing withuracil, and a puromycin inhibiting protein synthesis did not show anyeffect on cell viability. Based on these results, it has beendemonstrated that there is no significant relations between the abilityof antibiotics to enhance cell viability under hypoxic condition and theaction mechanism of antibiotics or the chemical structure ofantibiotics. Although efficacy of antibiotics to maintain cell viabilityunder hypoxic condition varies, effective concentration range ofantibiotics on enhancement of viability of human hepatoma cell line wasabout 0.1 to 1000 μg/ml. Meanwhile, trimetazidine which is known toenhance cell viability by increasing utilization of glucose under ahypoxic condition did not show positive results in the presentinvention.

Administration Route and Effective Dose

The pharmaceutical compositions comprising antibiotics of quinolones,quinones, aminoglycosides or chloramphenicol as active ingredients andpharmaceutically acceptable carriers can be administered by an injectionformula. As the injection formula, isotonic aqueous solution orsuspensions are preferred, which are sterilized and/or supplemented withpreservatives, stabilizers, wetting agents, emulsifiers, or salts forcontrolling osmotic pressure and/or buffers. In addition, they mayfurther comprise other therapeutically useful substances.

Upon using the therapeutic agents comprising antibiotics as activeingredients, the antibiotics are administered to an individual in anamount of administering to the individual infected with pathogenicmicroorganisms. Though the effective dose of an aminoglycosideantibiotic like geneticin is variable depending on the age, body weightof patient and progression of disease, it is preferable to administerparenterally 2 to 3 g/day to an average adult (body weight of 60 kg) ina single dose, which may be individualized by experience of the skilledin the art.

Acute Toxicity Test

All antibiotics used in the present invention, except for anaminoglycoside antibiotic of geneticin, were not subject to acutetoxicity tests since they have been clinically approved for theirsafety. In case of geneticin, after subcutaneously injecting geneticininto male C57BL/6 mice, dead mice were counted through 1-7 days. Theresults demonstrated that the therapeutic agent comprising geneticin isobviously a safe pharmaceutical drug in an effective dose range, in aview that LD₅₀ value is about 3800 mg/kg.

As clearly illustrated and demonstrated above, the present inventionprovides therapeutic inhibiting apoptosis under ischemic condition,comprise antibiotics of quinolones, quinones, aminoglycosides orchloramphenicol as active ingredients. The therapeutic agents improvesthe viability of cells under hypoxic and hypoglycemic condition,assuring that they can be clinically applied for ischemia-associateddiseases such as myocardial infarction and cerebral infarction, andbiotechnology as well.

Various modifications of the invention in addition to those shown anddescribed herein will be apparent to those skilled in the art from theforegoing description. Such modifications are also intended to fallwithin the scope of the appended claims.

What is claimed is:
 1. A method for inhibiting apoptosis under ischemicconditions in an individual in need of such inhibition, the methodcomprising administering to the individual an effective amount toinhibit apoptosis under ischemic conditions of a composition comprisingat least one quinolone antibiotic substance and a pharmaceuticallyacceptable carrier.
 2. The method of claim 1, wherein the quinoloneantibiotic substance is selected from the group consisting oflevofloxacin, ofloxacin and ciprofloxacin.
 3. A method for inhibitingapoptosis under ischemic conditions in an individual in need of suchinhibition, the method comprising administering to the individual aneffective amount to inhibit apoptosis under ischemic conditions of acomposition comprising at least one aminoglycoside antibiotic substanceand a pharmaceutically acceptable carrier.
 4. The method of claim 1,wherein the at least one aminoglycoside antibiotic substance is selectedfrom the group consisting of geneticin, neomycin and gentamycin.
 5. Amethod for inhibiting apoptosis under ischemic conditions in anindividual in need of such inhibition, the method comprisingadministering to the individual an effective amount to inhibit apoptosisunder ischemic conditions of a composition comprising chloramphenicoland a pharmaceutically acceptable carrier.